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Broad-led Scientists Find New Cas9 Enzyme for In Vivo CRISPR Genome Editing

NEW YORK (GenomeWeb) – The Cas9 nuclease from Staphylococcus aureus can be used to edit genomes as efficiently as Cas9 from Streptococcus pyogenes but at a smaller size, enabling CRISPR methods for in vivo applications that could eventually include therapeutic use, researchers reported today in a study published in Nature.

Scientists from the Broad Institute, the Massachusetts Institute of Technology, and the National Center for Biotechnology Information of the National Institutes of Health used comparative techniques to search metagenomic sequencing databases for a Cas9 protein suitable for mammalian in vivo CRISPR genome editing, yielding an enzyme from S. aureus that can be packed into a viral vector. They demonstrated its ability to induce a measurable change in physiology by targeting the PCSK9 gene, where a loss of function is associated with improved cardiovascular health in humans. They said they were able to successfully target the gene for indel mutations and reduced cholesterol levels in the mice.

"This study highlights the power of using comparative genome analysis to expand the CRISPR-Cas9 toolbox," Broad Institute core member Feng Zhang, a senior author on the study, said in a statement. "This new Cas9 provides a scaffold to expand our Cas9 repertoire, and helps us create better models of disease, identify mechanisms, and develop new treatments."

The S. aureus Cas9 (SaCas9) is 25 percent smaller than the S. pyogenes Cas9 enzyme (SpCas9) that has already been proven to work in editing higher order genomes. This means the SaCas9 can be packed into a single adeno-associated virus (AAV) for delivery into living cells.

AAV is a promising candidate vector for in vivo applications of CRISPR/Cas9, since it is not known to cause human disease and has been approved for clinical use in Europe. However, its payload capacity is too small to fit the DNA construct packing both the SpCas9 enzyme and the other components required for CRISPR genome editing.

Prior attempts to deliver CRISPR/Cas9 systems in vivo required multiple AAVs, so the researchers looked for a bacterium with a shorter Cas9 coding sequence than S. pyogenes. Phylogenetic analysis of metagenomic databases turned up six candidates, representative of all the bacterial species that produce the Cas9 protein. The sequence coding for Cas9 was sufficiently short in all six, but SaCas9 was the only enzyme that demonstrated DNA cleaving ability in mammalian cells comparable to that of SpCas9.

Though the S. aureus enzyme is distantly related to the S. pyogenes enzyme, relatively speaking, it also had comparably low levels of off-target effects across the entire genome, as determined by a method to detect double-stranded breaks and other undesirable damage known as direct in situ Breaks Labeling, Enrichment on streptavidin and next-generation Sequencing (BLESS). BLESS testing was not done in vivo, but in cell lines similar to the cells targeted later on.

"We don't want to claim it's better, but it's comparable," to the S. pyogenes CRISPR system, Le Cong, a co-lead author of the study, told GenomeWeb.

To show that SaCas9 could edit genomes in vivo, the researchers targeted PCSK9 for disruption in adult mice. The loss of PCSK9 in humans has been associated with the reduced risk of cardiovascular disease and lower levels of LDL cholesterol, making it a promising drug target. In a mouse model, the team observed almost complete depletion of the PCSK9 convertase protein in blood one week after administrating the CRISPR/SaCas9-packing AAVs. The mice showed a 40 percent decrease in total cholesterol and did not have overt signs of adverse side effects such as inflammation or immune response.

The scientists said that SaCas9 could be engineered to allow the targeted control of gene expression in vivo, which researchers could use to better understand transcriptional and epigenetic regulation in the cell. Gene promoter, gene suppressor, and reporter proteins have all been fused to non-cleaving SpCas9 enzymes to create important research tools.

Genome editing in vivo could also allow new types of animal studies that researchers couldn't do before, Cong said. While some neurological and metabolic diseases like Alzheimer's or type 2 diabetes have genetic components, they're not inborn diseases. "To best recapitulate or study them in animal models, we need to understand what's happening during adulthood," Cong said.

"In vivo gene editing allows you to perturb the system in adults to really understand the mechanisms of how the disease happens and might allow you to develop therapeutic approaches to apply in adulthood," he added.

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